Our research program is focused on the analysis of the three-dimensional structures of macromolecular assemblies using high resolution electron microscopy. Members of our group work on different, yet, complementary aspects of structural analysis. These include electron crystallographic studies of two-dimensional protein crystals, "single particle" approaches to analyze the structures of protein complexes and determination of the structures of large subcellular assemblies using electron tomography. A significant fraction of our research effort is devoted to developing and implementing novel methods for specimen preparation, high throughput data acquisition and computational analysis. Research overview 2002-2003: Highlights from selected projects are summarized below: (i) Membrane proteins: We are exploring the structural basis underlying the transport of ions and solutes across biological membranes using high resolution electron crystallography at resolutions in the 3 to 6 range. Recent projects have included studies of the mechanism of proton pumping by bacteriorhodopsin, mechanism of oxalate transport by the 12-helix oxalate transporter. In both cases we have deduced the mechanism of transport by determining sructures of the proteins in two different conformations with accessibility to alternate sides of the bilayer membrane. (ii) Signal transduction mechanisms and structure of molecular machines: A variety of projects are underway to explore fundamental mechanisms underlying signaling mechanisms in prokaryotic and eukaryotic cells. For example, we are using electron tomography and single particle electron microscopy to analyze the spatial architecture of the signaling apparatus used by bacteria to sense and respond to extracellular ligands. We are studying the molecular structure of multiprotein complexes involved in cellular metabolism and the large complexes that serve as rotary machines to support motion of bacterial flagella. We are investigating changes in mitochondrial architecture that are associated with apoptosis mediated by proteins such as Bax. (iii) Cell-cell interactions: Several projects are underway to explore fundamental mechanisms involved in cell-cell contact and ensuing interactions, such as studies of the entry of HIV into target cells, studies of the immunological synapse formed at the contact region between dendritic cells and T-cells, and studies of remodeling of the surfaces of peripheral blood T-lymphocytes in response to their detection of chemokines. (iv) We have continued our development of tools for automation in data collection and image processing with implementation of new methods for docking X-ray structures into EM maps and with the use of improved CCD detectors.